MYPT1-PP1β phosphatase negatively regulates both chromatin landscape and co-activator recruitment for beige adipogenesis

Protein kinase A promotes beige adipogenesis downstream from β-adrenergic receptor signaling by phosphorylating proteins, including histone H3 lysine 9 (H3K9) demethylase JMJD1A. To ensure homeostasis, this process needs to be reversible however, this step is not well understood. We show that myosin phosphatase target subunit 1- protein phosphatase 1β (MYPT1-PP1β) phosphatase activity is inhibited via PKA-dependent phosphorylation, which increases phosphorylated JMJD1A and beige adipogenesis. Mechanistically, MYPT1-PP1β depletion results in JMJD1A-mediated H3K9 demethylation and activation of the Ucp1 enhancer/promoter regions. Interestingly, MYPT1-PP1β also dephosphorylates myosin light chain which regulates actomyosin tension-mediated activation of YAP/TAZ which directly stimulates Ucp1 gene expression. Pre-adipocyte specific Mypt1 deficiency increases cold tolerance with higher Ucp1 levels in subcutaneous white adipose tissues compared to control mice, confirming this regulatory mechanism in vivo. Thus, we have uncovered regulatory cross-talk involved in beige adipogenesis that coordinates epigenetic regulation with direct activation of the mechano-sensitive YAP/TAZ transcriptional co-activators.

percentage should be included. 4. The interpretation of the oil-red O experiment is puzzling. It was not fully explained in the text. 5. In Figs.5e, 5f, 5g, the number of biological replicates appears to be small. 6. Provide biological replicates and mice information for all experiments. 7. The western blot in Figs. 1d and 4g were performed individually, suggesting either inconsistent or incomparable. These blots should be reperformed in the same blot. 8. In place of bar graphs, small datasets show the full data as univariate scatter plots that also show mean and standard deviation (Fig. 1i,1j,1k,2e,2i,3d,3h,3i).
Reviewer #2 (Remarks to the Author): The manuscript titled "MYPT1-PP1B phosphatase negatively regulates both chromatin landscape and co-activator recruitment to influence gene expression during beige adipogenesis" by Takahashi et al reported a previously unknown regulator MYPT1-PP1B in beige fat biology. The group has previously reported that a histone H3 lysine 9 (H3K9) demethylase JMJD1A plays an important role regulating beige fat function, and is activated through phosphorylation at serine 265 by protein kinase A (Nature Communications 2015, Abe et al. and Nature Communications 2018 Abe et al). Current study is a nice follow-up to reveal the identity of the phosphatase that is responsible for the dephosphorylation of s265 in JMJD1A in beige fat. The unbiased experimental approach was well controlled and presented data were convincing. In addition, the author further uncovered an interesting connection between MYPT1-PP1B to myosin regulatory light chain (RLC) regulated pathway, which mediates beige fat function through TAZ/YAP. Even though this is continuation of previous study, the novelty and significance of the present study is evident.
The manuscript is well written and most of the results are of high quality, strongly supporting proposed hypothesis. In particular, the authors demonstrated that general adipogenesis is not affected by MYPT1-PP1B, rather its effects are specifically affecting thermogenic function. This separates current study from many other reports that claim protein X or Y are thermogenic fat regulators but most likely they just affect fat function in general.
This reviewer does have some comments/suggestions for data shown in figure 5, in particular the data using Mypt1+/flox;pdgfra-cre mice.
As of right now, there is no perfect Cre line for general preadipocytes, nor for beige fat precursor. PDGFRA-CRE has been used as preadipocyte cre line in many studies, but its caveats were well documented. Indeed, the fact that Mypt1flox/flox;pdgfra-cre mice die embryonically suggests that MYPT1 in other cell types in addition to preadipocyte may play an essential role in development. The drastic phenotype shown in fig5E and fig5g from heterogeneous Mypt1 in PDGFRA-lineage is a bit complicated to interpret. The most responsive organs upon acute cold exposure are skeletal muscle which regulates shivering and classical brown fat which will be immediately activated upon cold. In comparison, beige adipogenesis within subcutaneous fat upon cold exposure, the full extent of activation may take a long time and involve tissue remodeling, including new precursor for beige adipocytes to differentiate. The fact that after only 8 hrs, there is body weight change and subcutaneous adipose tissue mass change and body temperature change, it is unlikely caused by half dose of mypt1 in just beige precursors. Further exploration should be carried out. Alternatively, the authors may consider to just remove this model from the current study, since there are several other in vivo models, which are better controlled with fewer caveats and also support the hypothesis.
Minor point: 1. In data availability section, the authors indicate that the RNA-seq transcriptome data and JMJD1A ChIP-seq data have been deposited at GEO but the accession number was listed as "GSEXXXXX". Customarily, most authors deposit these data as private data protected with a password, with public release set upon publication date of the manuscript. An actual GSE number should be included, and a password should be made available to reviewers upon request. Reply to major comment 1-1: We appreciate the valuable comment made by the reviewer. Regarding (i) Immunoblotting showed that UCP1 protein levels were significantly elevated in scWATs of mice injected with AAV-mCherry-2A-Cre compared to AAV-mCherry (Fig.   5b). AAV-mCherry-2A-Cre injection slightly decreased scWAT weight without affecting body weight (Fig. 5f). TOM20 was used as a mitochondrial loading control. n = 7 per group.

Fig. 5c
Expression of thermogenic genes in the scWAT of AAV-injected Mypt1 flox/flox mice (line 2) after 1 week of acclimation to 12 °C. Data are presented as mean ± SEM (n = 10 per group, left). P-values by paired t-test. NE-induced OCR normalized to tissue mass in scWAT AAV-injected Mypt1 flox/flox mice after 1 week of acclimation to 12 °C (right). Data are presented as the mean ± SEM (AAV-CMV-mCherry, n = 10; AAV-CMV-mCherry-2A-Cre, n = 12). P-values were calculated using Student's t-test.  (i) Immunoblotting showed that UCP1 protein levels in scWAT were significantly higher than those in control mice when Mypt1 +/flox ::Pdgfra-Cre mice were housed at 12 °C for one week (Fig. 5g). (ii) Histological analysis showed that Mypt1 +/flox ::Pdgfra-Cre mice had more UCP1positive multilocular beige adipocytes in scWAT than control mice at RT (Fig. 5i). (iii) qPCR analysis demonstrated that the expression of thermogenic gene mRNA was increased in the scWAT of Mypt1 +/flox ::Pdgfra-Cre mice even at RT (Fig. 5h). With regards to revised Supplementary Fig. 5i, results of an in vivo experiment using Mypt1 +/flox ::Adipoq-Cre mice, gene expression profiling, and histological studies were not performed because no thermogenic phenotypes were obtained, such as increased Ucp1 expression in scWATs or increased cold tolerance by acute cold exposure (4 °C).
These data suggest that JMJD1A does not contribute to the trans-differentiation of mature white adipocytes into beige adipocytes in scWAT.

Reply to major comment 1-2:
We appreciate your valuable comment. Therefore, we immunostained serial sections of scWAT from Mypt1 flox/flox mice injected with AAV-mCherry-2A-Cre with either UCP1 or mCherry antibody, separately, and examined whether mCherry-2A-Cre positive cells (i.e., MYPT1-depleted cells) overlapped with UCP1 positive cells. The results showed that most of the multilocular adipocytes colocalized with UCP1 and mCherry, whereas mCherry-negative, UCP1-positive beige adipocytes were hardly detected. This indicated that MYPT1 depletion promoted beige adipocyte formation in a cell-autonomous manner (Fig. 5d).

Reply to major comment 2-1:
We assume that the reviewer refers to H3K9me2 levels and not H3K4me2 levels, as the former is the substrate for JMJD1A. As shown in Fig. 1n in the original manuscript, simultaneous depletion of both MYPT1 (regulatory subunit) and PP1β (catalytic subunit) by siRNAs resulted in higher H3K9me2 levels in the thermogenic gene (i.e., Ucp1) in differentiated im-scWAT. However, it is unclear whether this is dependent on the H3K9me2 demethylation activity of JMJD1A.
Therefore, to determine whether this change in H3K9me2 is mediated by JMJD1A, we examined H3K9me2 levels on the Ucp1 gene enhancer by overexpression of a demethylation-defective JMJD1A mutant (H1120Y) or WT-JMJD1A. In im-scWAT overexpressing WT-human JMJD1A, H3K9me2 levels in the Ucp1 enhancer were reduced by Mypt1 knockdown (Fig. 3j). In contrast, in H1120Y-human JMJD1Aoverexpressing im-scWAT, H3K9me2 levels on the Ucp1 enhancer were high, and Mypt1 knockdown hardly reduced H3K9me2 levels. These results indicate that Ucp1 expression is regulated by the MYPT1-JMJD1A axis and that the decrease in H3K9me2 levels upon MYPT1 depletion is dependent on JMJD1A demethylation activity.

Reply to major comment 2-2:
We agree that the present study did not show the levels of H3K9me2 in scWATs of AAV-Cre-injected Mypt1 flox/flox mice or conditional Mypt1-deficient mice. However, it is technically difficult to see a decrease in H3K9me2 at 12 ºC for one week of cold stress in Mypt1 partially deficient mice ( Fig. 1 only for reviewers). This is because, as shown by UCP1 immunohistochemistry (Fig. 2 only for reviewers), the "beige-ing" of scWAT is much milder at 12 °C than at 8 °C.

Fig. 2 only for reviewers
Immunohistochemical analysis of scWAT from mice acclimated at 8 °C for two weeks (left) or 12 °C for one week (right). The scWAT was stained with UCP1 antibody.
In addition, scWAT is composed of various cell types, including white adipocytes, beige adipocytes, pre-adipocytes, immune cells, blood cells, and endothelial cells. A recent study reported that adipocytes account for only 50% of all cell types in scWAT (Cell Reports Roh et al. 2017). Therefore, it is not easy to see a specific decrease in H3K9me2 on thermogenic genes (e.g., Ucp1) by beige-ing because the affected cells comprise only a small fraction of the total cells present in adipose tissue.
We previously reported a slight but significant decrease in H3K9me2 on thermogenic genes in scWAT beige-ing under severe cold stress condition at 4 °C (Abe Y. et al., Nat Commun 2018; Fig. 1f, see below figure), but in this temperature, many more beige adipocytes were observed. The slight decrease in H3K9me2 in the tissue is due to heterogeneity of the scWAT population, with adipocytes (white and beige) accounting for only 50% of all scWAT cell types, as described above. In addition, beigeing decreases H3K9me2 in thermogenic genes (e.g., Ucp1) but not in other cell types, where H3K9me2 levels are likely high because thermogenic genes are not turned on.
ChIP-qPCR in scWAT from mice placed at 4°C for 1 week (n = 4). The data from Abe Y. et al., 2018 Nat Commun. H3K9me2, Fig. 1f Therefore, to evaluate a more homogeneous cell population, we first showed that Mypt1 depletion reduces H3K9me2 on Ucp1 gene enhancers and that the MYPT1-JMJD1A axis regulates H3K9me2 on the Ucp1 enhancers using SVF-derived cultured pre-adipocytes of scWATs. Next, we generated conditional knock-out mice to validate this concept. The knock-out phenotype was consistent with cultured scWAT showing increased thermogenic capacity due to MYPT1 depletion.  Unfortunately, we were unable to demonstrate changes in RLC phosphorylation in vivo under these conditions. RLC phosphorylation levels at 12°C may not be high enough to be detected with antibodies.

Major comment 4:
In this study, the authors assumed that Ucp1 mRNA expression (Figs. 2e,2h,2i,3d,3h,3l,4h) would be the marker of beige adipogenesis. Not only Ucp1 but also other thermogenic genes and beige marker genes should be investigated.
Reply to major comment 4: We appreciate your valuable comments. We have revised the manuscript to show the expression of not only Ucp1, but also other thermogenic genes, in the corresponding figures. These results strengthen our major conclusion that the MYPT1-JMJD1A axis regulates beige adipogenesis.   (PMID: 29657031, 29692364, 27568548). In this regard, the importance of this study would be further strengthened when they examine whether MYPT1 is involved in beige-to-white transition.
Reply to major comment 5: We thank the reviewer for valuable comments. Yes, PKA inhibits MYPT1/PP1 phosphatase activity via T694 phosphorylation of MYPT1. It is possible that MYPT1/PP1β turns on/off PKA signaling in the reverse process, beigeto-white transition. The original focus of this study was the role of MYPT1 in coldinduced beiging of scWAT and we have already included a very large set of data to investigate this process. Thus, we hope the reviewer would agree that this interesting additional possibility is a separate question that we think would be best examined in a future study.

Major comment 6:
The authors suggested that MYPT1 would play suppressive roles in preadipocytes, not in mature beige adipocytes, which was supported by Adipoq-Cre Mypt1 KO mice model  With regards to Adipoq-Cre::Mypt1 flox/flox mice, they do not exhibit thermogenic phenotype, suggesting that phospho-JMD1A does not promote trans-differentiation from white mature adipocytes to beige adipocytes.
In addition, as a preliminary experiment, primary cultured SVFs were prepared from scWATs of Mypt1 flox/flox mice and infected with adenovirus carrying Cre recombinase (Adeno-Cre) on the day before (Day 1; pre-adipocyte stage) or seven days after (Day 7; mature adipocyte stage) differentiation to eliminate MYPT1. After differentiation, adipocytes were harvested on day 8 and thermogenic gene expression was examined. The results (Fig. 3 only for reviewers) showed that the expression of thermogenic genes was higher in cells infected on day 1 than in those infected on day 7, suggesting that Mypt1 depletion does not affect the transcription of thermogenic genes in mature beige adipocytes, but only early in differentiation. We acknowledge that this is not a perfect experiment, and there are limitations in the interpretation; however, the results are consistent with the model and it will require significant future studies to clarify these points.
The following sentences are added to each figure legend. "d, e, f, i, j, k, l, n Representative of three (e, f, i, j, k, l) or two (d, n) independent experiments. Data are presented as the mean ± SEM of three technical replicates in i, j, k, and n. Representative data are presented in Supplementary Table 1. n Unpaired two-tailed Student's t-test. i, j, and k One-way ANOVA with Tukey's multiple comparisons test." (Page 57 to 58, Fig. 1 legend of revised manuscript).
"e, f, g, i Representative of three (e, i) or two (f, g) independent experiments. Data are mean ± SEM of three technical replicates in e and i. e, h, i One-way ANOVA with Tukey's multiple comparisons test." (Page 59, Fig. 2 legend of revised manuscript). Reply to comment 2: To examine the relationship between the binding sites of JMJD1A and gene expression, we defined JMJD1A-regulated genes as those that met the following criteria: expression was induced more than 1.5-fold or less than 1/1.5fold and localized within 50 kb from JMJD1A binding sites, as described under "Methods". The score for each gene (JMJD1A binding) was defined as the sum of the scores of the peaks located in the ±50 kb region around its TSS. A higher score indicated stronger binding of JMJD1A to the promoter or enhancer region of the gene of interest.
We used a setting of 50 kb according to the following published papers: (1)  (3) Epigenetics & Chromatin, 2020 [10.1186/s13072-020-0327-0]. (Figs. 1b, 1c), more details such as peptide numbers and protein match percentage should be included Reply to minor comment 3: According to the comment, a table showing peptide numbers and protein match rates of the MS/MS data has been included as "Supplemental Data 1" in the revised manuscript.

Minor comment 4: The interpretation of the oil-red O experiment is puzzling. It was not fully explained in the text.
Reply to minor comment 4: We thank the reviewer for this important comment. This is related to the following comment by reviewer #2: "This separates current study from many other reports that claim protein X or Y are thermogenic fat regulators but most likely they just affect fat function in general." To show that Mypt1 knockdown (or other settings) did not affect lipid accumulation (i.e., general adipogenesis), but did affect beige adipogenesis (thermogenic gene expression), we presented the results of Oil Red O staining, but this point was not clearly explained in the original paper. Therefore, we have added the following sentence to the revised manuscript: "Oil red O staining showed that lipid accumulation (i.e., general adipogenesis) is not affected by MYPT1 depletion (Fig. 1i, inset), indicating MYPT1 regulates specific thermogenic genes during adipogenesis." (Page 8, lines 9-11) Minor comment 5: In Figs.5e, 5f, 5g, the number of biological replicates appears to be small.

Reply to minor comment 5:
We agree with the reviewer's concern. Shown in Fig. 5f and 5g in the original manuscript (acute cold exposure), there were three biological replicates; therefore, we omitted these experiments. Alternatively, we analyzed Mypt1 +/flox ::Pdgfra-Cre mice after chronic (1 week) cold exposure at 12 °C or RT. After cold exposure, UCP1 protein levels in scWAT were significantly higher than those in control mice (Fig. 5g in the revised manuscript). In addition, Mypt1 +/flox ::Pdgfra-Cre mice showed higher expression of thermogenic genes and UCP1-positive multilocular beige adipocytes than control mice at RT (Fig 5h, 5i in the revised manuscript). These results indicate that MYPT1 depletion in pre-adipocytes promotes beige adipogenesis.
The acute cold tolerance test in the original Fig. 5e was performed under RT, where beige adipogenesis was significantly promoted in Mypt1 +/flox ::Pdgfra-Cre mice compared to that in control mice. Body temperature change in control mice, although the number of biological replicates was small, was comparable to that in control mice in Supplementary Fig. 5j. In contrast, knockout mice (n = 6) were more resistant to cold stress compared to control mice, shown in Fig. 5i. Thus, we considered that the data are promising and have kept this data in Supplementary Fig. 5q in the revised manuscript.
In addition, we included metabolic data from mice of this genotype fed a high-fat diet (HFD) to confirm the higher energy expenditure phenotype. HFD feeding resulted in higher body weight gain in the control (Mypt1 +/flox ) group, while the Mypt1 +/flox ::Pdgfra-Cre mice (male, n = 20-22), which exhibited less body weight gain in comparison (Fig. 5j). These mice had improved glucose tolerance and reduced plasma insulin levels, showing an improved metabolic phenotype when challenged with HFD (Fig. 5k, 5i, and Supplementary Fig. 5r). These data alternatively support a higher thermogenic/energy consumption phenotype in Mypt1 +/flox ::Pdgfra-Cre mice.
We have removed the original Fig. 5f and 5g from the revised manuscript. We hope that the reviewer agrees that these additions and removals increased the overall quality of the study presented here.
The following sentences were added.
Expression of thermogenic genes was also increased in the scWAT of Mypt1 +/flox ::Pdgfra-Cre mice, even in RT housing (Fig. 5h). Histological analysis showed that Mypt1 +/flox ::Pdgfra-Cre mice had more UCP1-positive multilocular beige adipocytes in scWAT than did control mice acclimated at RT (Fig. 5i)." (Page 17, lines 5-11) "In addition, HFD feeding at RT resulted in higher body weight gain in control mice, while Mypt1 +/flox ::Pdgfra-Cre mice exhibited reduced body weight gain in comparison (Fig. 5j). Furthermore, Mypt1 +/flox ::Pdgfra-Cre mice showed improved glucose tolerance and lower serum insulin levels under fasting conditions or after glucose injection compared with control mice (Fig. 5k, 5l,   Supplementary Fig. 5r), indicating that MYPT1 depletion is associated with a higher energy consumption phenotype and improved glucose metabolism. These    Reply to minor comment 6: As in our response to minor comment 1, we have provided the number of biological replicates for all experiments in the figure legends in the revised manuscript. In addition, we provide a

Reply to minor comment 7:
We repeated the co-immunoprecipitation analysis of Fig. 1d and confirmed an interaction between MYPT1 and JMJD1A. We also repeated the P-JMJD1A immunoblot analysis of Fig. 4g and confirmed that ISO-induced JMJD1A phosphorylation was markedly decreased in T696A-MYPT1 cells. The new data are shown in Fig. 1d and 4g in the revised manuscript.  In addition, we want to emphasize that the original data shown in Fig. 1d and 4g were not performed individually, but on the same blots ( Fig. 4 and Fig. 5

only for reviewers).
Irrelevant samples on the gels were eliminated. We apologize for the lack of uncropped images, causing ambiguity, in the initial submission.

Reply to major comment
We appreciate reviewer's thorough understanding of our manuscript.
Firstly, we will address the concern "In comparison, beige adipogenesis within subcutaneous fat upon cold exposure, the full extent of activation may take a long time We would like to emphasize that we do not think that beige-ing of Mypt1 flox/+ mice occurred within 8 h of acute cold exposure, but through rearing at RT. We also need to emphasize that the data in the original Fig. 5g show no significant change in body weight or subcutaneous adipose tissue mass after 8 h of cold exposure between the two groups ; therefore, beige-ing did not occur within this short time period.
As the reviewer may know, RT is lower than the thermoneutral temperature, 30 °C, so an intermediate cold stimulus is present. Beige-ing occurs via sympathetic nerve activity. Beige-ing was promoted in scWATs of mice reared at RT; Pdgfr-Cre::Mypt1 flox/+ mice showed a higher expression of thermogenic genes associated with beige-ing than the control group at RT, as represented by Ucp1, Cpt1b, and Ppara (P < 0.05) (Fig. 5h). Furthermore, Mypt1 +/flox ::Pdgfra-Cre mice had more UCP1-positive multilocular beige adipocytes in scWAT than control mice at RT (Fig. 5i).
It has been reported that mice with advanced beige-ing become resistant to acute cold (Ikeda, K. et al. Nat Med 23, 1454-1465, 2017 In addition, as in our response to minor comment 5 from reviewer #1, we would like to provide additional metabolic data regarding Mypt1 flox/+ ::Pdgfra-Cre mice that have reduced body weight gain, better glucose tolerance, and lower plasma insulin upon increased glucose challenging. These data alternatively support a higher thermogenic/energy consumption phenotype in Mypt1 +/flox ::Pdgfra-Cre mice (Figs. 5j,   5k, and 5l). We hope that we could provide a satisfactory response to your constructive criticism.

Minor point 1:
In data availability section, the authors indicate that the RNA-seq transcriptome data and JMJD1A ChIP-seq data have been deposited at GEO but the accession number was listed as "GSEXXXXX". Customarily, most authors deposit these data as private data protected with a password, with public release set upon publication date of the manuscript. An actual GSE number should be included, and a password should be made available to reviewers upon request.
Reply to minor point 1: We apologize that the accession numbers of RNA-seq transcriptome data and JMJD1A ChIP-seq data were not provided in the original manuscript. The GSE number (GSE202506) has been included in the revised manuscript. A password for accessing the data will be provided to reviewers upon request.
The following sentence was added.